Study On Antifungal And Antiviral Effects And Mechanism Of Several Plant-derived Active Substances

Plant-derived active substances are major part in natural pesticides development.In this study,the methods of screening antiviral substances are explored and optimized,and the suppression effects of previously reported plant-derived substances,which may have antiviral or antifungal activity,on Alternaria alternata and Tobacco mosaic virus(TMV)are investigated.The main findings are as follows:1.Using Nicotiana benthamiana(N.benthamiana)and several antiviral agents as experimental materials,the agents were injected into five points of the tobacco leaf or the whole leaf.The TMV-GFP was then inoculated.The area of the fluorescent spot and the spots number of different agents treated-leaves were observed under UV lamps at two days post inoculation(dpi).The results showed that the fluorescence intensity of each treatment was lower than that of the control.The statistical results of the fluorescent spot were similar to those of the ELISA,which indicated that the fluorescence intensity could reflect the virus accumulation level in leaves.And the evaluation system for several antiviral agents indoors was nearly the same with that in the fields,indicating that the two screening methods established in this study is available for the evaluation and screening of antiviral agents.Compared with local lesion and leaf disc method,the system can not only quickly evaluate the activity of antiviral agents,but also can intuitively reflect the role of agents in the process of virus diffusion and replication.It has the advantages of simple operation,intuitive phenomenon and short cycle.2.TMV was used as the target for the antiviral activity assay of 15 kinds of plant material.After screening combined with defense-related enzyme activity assay,two kinds of active agents(ursolic acid and 4-methoxycoumarin),which could reduce the initial infection concentration of virus,were obtained.The results showed that ursolic acid and 4-methoxycoumarin could increase the activity of SOD,PPO,POD and CAT and reduced the content of peroxide in N.benthamiana leaves to some extend;In the early stage of virus infection,ursolic acid and 4 – methoxycoumarin could reduce the degree of membrane lipid peroxidation of plant cells and cell membrane damage,and improve the PR1,NPR1 and PR2 and other defense response-related gene expression,promote disease-related protein synthesis,thereby reducing the initial infection content of virus,with the role of protection and delayed disease development.In vitro studies showed that 1 h after the two active substances mixed with TMV,the virus particles occurred to be aggregated with solvent-treated virus dispersed,The accumulation of ursolic acid treated virus particles was more obvious than that of 4-methoxycoumarin.After the treatment of the two substances,the virus sap was frictionally inoculated to N.benthamiana,the number and intensity of fluorescent spots was decreased,indicating that the two substances in vitro directly affected the virus particles and reduced the virus infection activity or in vivo replication level.These results mean that the virus may be passivated by ursolic acid and 4-methoxycoumarin.3.The antifungal activities of 15 plant-derived substances were determined by plate antifungal method.It was found that clove oil and eugenol had good antifungal effect on Alternaria alternata.The germination of spores of Alternaria alternata could be inhibited with 4.13% clove oil,and the growth of mycelium was significantly reduced with 0.52% clove oil.Further studies have found that clove oil could destroy the cell structure of Alternaria alternata,increase plasma membrane permeability,lead the cell inclusions exosmosis,and increase the degree of membrane lipid peroxidation and the content of malonaldehyde,finally result in mycelial cell death and inhibition of pathogen growth and reproduction.